Determining the relationship between Sox9 and Collagen XVIII in maintaining intestinal stem cell identity

 描述(申请人提供)：肠道干细胞(ISCs)位于隐窝的底部，位于含有支持性Paneth细胞和胸骨周围肌成纤维细胞的壁龛内。细胞外基质(ECM)是生态位中研究较少的成分，也与ISCs密切相关，并可能影响ISC的行为，就像在其他组织中一样。尽管从本质上控制ISC维护的关键机制开始被识别，但关于外部因素(如ECM)如何影响ISC的信息很少。转录因子Sox9在小肠隐窝内以不同方式标记ISCs，这是正常ISC所必需的 功能，并已被证明是必要的适当的上皮细胞增殖。Sox9也与细胞外基质的调节有关，在缺乏Sox9(Sox9-KO)的肠上皮中，XVIII型胶原A型(Col18a1)表达下调。有趣的是，Col18a1具有结合Wnt的能力，Wnt是一种已知的Sox9和ISC增殖调节因子。Sox9、Col18a1和Wnt信号之间的关联表明，Sox9可能是ISC生态位内动态互惠的驱动因素，使ISCs与周围的ECM相互作用。因此，这一提议的中心假设是，Sox9通过在小肠隐窝中创建Wnt梯度来驱动Col18a1的产生，以维持ISC的身份。为了验证这一假设，我们将首先通过对野生型肠隐窝上皮和支持胸骨周围肌成纤维细胞的单细胞RNA测序来确定Col18a1在ISC生态位中的细胞来源。收集的数据将为未来的研究提供关于Col18a1的细胞起源以及任何其他差异表达基因的信息。然后，我们将通过无细胞野生型和Sox9-KO ECM的质谱学来确定Sox9对生态位ECM中Col18a1蛋白的影响。Western blotts和免疫组织化学将确定在Sox9-KO ECM中Col18a1的丰度和位置以及任何异常表达模式。最后，我们将通过慢病毒转导培养的ISCs中的Col18a1 siRNA来沉默Col18a1的表达，从而确定Col18a1对ISC功能的影响。确定Sox9和Col18a1之间的关系对于了解ISC/ECM在动态平衡、损伤后再生和疾病过程中的动态以及开发新的肠道组织工程策略至关重要。

 DESCRIPTION (provided by applicant): Intestinal stem cells (ISCs) reside at the base of the crypt, within a niche that contains supportive Paneth cells and pericryptal myofibroblasts. The extracellular matrix (ECM), an understudied component of the niche, is also intimately associated with ISCs and may be influential on ISC behavior, as it is in other tissues. Although key mechanisms that intrinsically control ISC maintenance are starting to be identified, little information exists on how extrinsic factors, such as the ECM, impact ISCs. The transcription factor Sox9 differentially marks ISCs within the small intestinal crypt, is required for proper ISC function, and has been shown to be essential for proper epithelial proliferation. Sox9 has also been associated with regulation of ECM, with Collagen XVIII Type A (Col18a1) down-regulated in intestinal epithelium lacking Sox9 (Sox9-KO). Interestingly, Col18a1 has the ability to bind Wnt, a known regulator of Sox9 and ISC proliferation. Association between Sox9, Col18a1 and Wnt signaling suggests that Sox9 may be a driver of dynamic reciprocity within the ISC niche, allowing ISCs to interact with the surrounding ECM. Therefore, the central hypothesis for this proposal is that Sox9 drives the production of Col18a1 to maintain ISC identity by creating a Wnt gradient in the small intestinal crypt. To test this hypothesis, we will first determine the cellular origin of Col18a1 within the ISC niche by single-cell RNA sequencing of wild type intestinal crypt epithelium and supportive pericryptal myofibroblasts. Data collected will provide information about the cellular origin of Col18a1 as well as any other differentially expressed genes for future studies. We will then determine the effect of Sox9 on Col18a1 proteins within the niche ECM by mass spectrometry of acellular wild type and Sox9-KO ECM. Western blots and immunohistochemistry will identify abundance and location of Col18a1 and any aberrant expression patterns in Sox9-KO ECM. Finally, we will determine the influence of Col18a1 on ISC function by silencing Col18a1 expression by lentiviral transduction of Col18a1 siRNA in cultured ISCs. Identifying the relationship between Sox9 and Col18a1 is critical for understating ISC/ECM dynamics during homeostasis, regeneration following injury, and disease, as well as developing novel intestinal tissue engineering strategies.